Germline stem cells (GSCs) produce sperm and egg, and the proper regulation of these stem cells is therefore essential for reproductive health. GSCs are regulated by short-range signaling from nearby niche cells that keep GSCs in a self-renewing state while allowing daughter cells to begin differentiation. However, recent studies have demonstrated that GSCs are also highly sensitive to whole-animal physiological cues, including nutrition, mating status, and age. While the short-range signals exchanged between GSCs and the niche are well characterized, the molecular mechanisms underlying long-range regulation of GSCs are not well understood. The goal of this study is to systematically identify and characterize new circulating factors that influence GSC proliferation. The proposed experiments will be conducted in Drosophila melanogaster, an established model system for GSC cell biology, possessing many parallels to vertebrate stem cell systems, and where it is uniquely possible to conduct genome-wide, in vivo genetic screens for factors released by specific organs that influence adult stem cell behavior. In Specific Aim 1, new tools will be applied to Drosophila GSCs to directly visualize the progression of the cell cycle, as well as the dynamics of local signaling pathway activity between GSCs and the niche. These techniques will overcome previous technical obstacles to monitoring GSC proliferation rate and signaling pathway activity, and will be used to test the effects of high- and low-nutrition on local GSC/niche signaling and cell cycling. Specific Aim 2 will test whether any of the known ligands for the 10 well-characterized, highly conserved animal signaling pathways are involved in tissue-extrinsic regulation of GSCs. Several of these factors have recently been implicated in a variety of inter-organ communication processes, but their roles in regulating adult stem cells such as GSCs have not been previously addressed. Each of these secreted ligands will be knocked down specifically in the fat body (the Drosophila analog of adipose tissue), muscles, gut, and brain, and the effects on GSC proliferation and local signaling pathway activity will be tested. Specific Aim 3 is a systematic, tissue-specific RNAi screen to identify novel organ-secreted factors influence GSC proliferation. All of the secreted factors in the Drosophila genome (n = ~800) will be knocked down in the same four tissues as in Aim 2, and effects on GSC proliferation will be screened. All of the new genes identified will then be tested to see whether they also influence the behavior of additional adult stem cell populations, or whether their effect are GSC-specific. Together, these studies provide insight into how adult stem cell populations integrate and respond to circulating signals from exogenous sources, a process with potential implications for such human health issues as spermatogenesis, polycystic ovarian syndrome, cancer risk, and systemic responses to nutrition.